Prokaryotic Expression of GA Module-containing Protein in Mycoplasma synoviae and Establishment of Indirect ELISA Method
[Objective]GA module-containing protein of Mycoplasma synoviae(MS)was expressed in vitro.A method for detecting MS antibody was established for serological detection and monitoring of MS antibody level.[Method]The long-chain conserved domain of GA module-containing protein in MS was analyzed and screened,and the recombinant plasmid pET30a-△GA-L was synthesized and transformed into Escherichia coli BL21(DE3)competent cells for induced expression,and the expression conditions were optimized.The expression of recombinant protein △GA-L was detected by SDS-PAGE.The recombinant protein △GA-L was purified,and identified by Western blotting.The purified recombinant protein △GA-L was used as the coating antigen to establish an indirect ELISA method for detection of MS antibody.The response conditions were optimized,the critical value was determined,the specificity,sensitivity and repeatability were tested,and clinical samples were tested.[Result]The molecular weight of the recombinant protein △GA-L was 45.7 ku,and the optimal expression conditions were 25 ℃ and 0.2 mmol/L IPTG induced expression for 5 h,and the expression was in the form of inclusion bodies.Western blotting result showed that the recombinant protein △GA-L could react specifically with MS antibody.An indirect ELISA method for MS antibody detection was established with △GA-L antigen coating concentration of 0.5 µg/mL,primary antibody dilution ratio of 1∶400 and secondary antibody dilution ratio of 1∶12 000 as the optimal conditions,and the critical positive value was 0.283.The established method had strong specificity,high sensitivity and high stability.The total coincidence rate was 96％compared with commercial MS antibody detection kit.[Conclusion]In this study,the MS recombinant protein △GA-L was successfully expressed,and the established indirect ELISA method for MS antibody detection had great specificity,sensitivity and repeatability,providing an effective and fast method for antibody detection of MS.
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